More people in the U.S. (160,340 per year) die of lung cancer than of prostate, breast, and colon cancer combined. The most common type of lung cancer, non-small-cell lung cancer (NSCLC), accounts for 75% of all lung cancers. Regrettably, 85% of all patients diagnosed with NSCLC eventually die of the disease within 5 years, due to micro-metastasis and relapse. Currently, over 12 million cancer survivors reside in the U.S., of which only 3.3% (about 400,000) are lung cancer survivors. Improved methods in effective cancer treatment are urgently needed and could greatly benefit not only lung cancer survivors, but also those of other cancer types. This project will develop a platform technology for exosome formulations, using bovine-milk-derived exosomes, of both lipophilic and hydrophilic drugs. Specifically, we will prepare exosome formulations of a standard chemotherapeutic (chemo) drug, paclitaxel (PAC), and a natural compound with chemopreventive and therapeutic potential, withaferin A (WFA), and examine their ability to prevent and treat cancer. We will focus on lung cancer and will seek to enhance the oral bioavailability of these drugs and reduce their required doses. Natural compounds often suffer from oral bioavailability issues, despite high doses. On the other hand, chemotherapeutic (chemo) drugs given intravenously are accompanied by undesirable large spikes in blood levels. Decades of work with liposomal and polymeric nanoparticle formulations have rendered only a handful of effective drugs for use in the clinics due to inherent limitations. In this application e will optimize exosome isolation and exosome formulations with potent therapeutic activity, and determine their therapeutic efficacy and potential toxicity. Production of the bovine milk-derived exosomes will be scalable, economically feasible, and highly cost effective. The exosomal drug delivery approach has the potential to change the current practice of cancer treatment by challenging the existing paradigm involving high doses of toxic chemo drugs. We hypothesize that exosome formulations of PAC and WFA administered orally will be more effective than respective free drugs (without exosome formulation), and that the formulation will reduce the required effective dose, thus minimizing or eliminating toxicity. The specific aims are to (1) optimize exosome isolation/purification from bovine milk, maximize drug loading of PAC and WFA, and determine stability of the exosome formulations; and (2) determine the efficacy of exosome formulations of PAC and WFA, in cell culture and in a nude mouse xenograft model, against human lung cancer cells, and also determine potential toxicities in a mouse model. Future efforts will optimize and determine the efficacy of nano spray-dried exosome formulations of PAC and WFA and other compounds, including siRNAs, determine their long-term stability, test formulations of PAC and WFA, both individually and in combination, against drug-sensitive, drug-resistant and metastatic lung cancer cells, using orthotopic tumor xenograft nude mouse models.